A DNA microarray allows the analysis of transcriptional expression of thousands of genes in a single assay (see, e.g., Ramsay (1998) Nature Biotechnol. 16:40–44; Marshall & Hodgson (1998) Nature Biotechnol. 16:27–31; Lashkari et al. (1997) Proc. Natl. Acad. Sci. (USA) 94:130–157; DeRisi et al. (1997) Science 278:680–6). The characterization of global cellular gene expression has many uses, including pathway dissection (see, e.g., Roberts et al. (2000) Science 287:873–80), classification of clinical samples (see, e.g., Khan et al. (1998) Cancer Res. 58:5009–13), drug evaluation (see, e.g., Hughes et al. (2000) Cell 102:190–26), and discovery of gene functions (see, e.g., Chu et al. (1998) Science 282:699–705).
In DNA microarray analyses, the identity of genes expressed in a sample and their levels of expression is determined by measuring the level of hybridization of nucleic acid “targets” to nucleic acid “probes” on the DNA microarray. A target sequence can be a messenger RNA (mRNA) present in the sample. More commonly, the target used is a nucleic acid molecule that is derived from the MRNA molecule and that has a sequence that is identical to, or complementary to, all, or a portion, of the mRNA molecule. A probe is a nucleic acid molecule on the DNA microarray that is complementary to a target.
Ideally, targets derived from MRNA molecules possess the following properties that facilitate their use in DNA microarray analyses. First, they should be representative of the mRNA population present in a sample. All, or substantially all, of the sequences in the mRNA population should be represented in the target sequence population used to screen the DNA microarray. Second, the relative abundance of each target sequence should be the same as the relative abundance of its corresponding mRNA in the sample. Third, each target should be present above a minimally detectable concentration to reliably measure its expression in the original samples. Fourth, the targets used to screen a DNA array should selectively hybridize to complementary nucleic acid molecules, and not hybridize to a significant extent to non-complementary nucleic acid molecules, immobilized on the DNA array or to the array surface surrounding the immobilized DNA.
The standard protocols for producing target RNA that can be used for DNA microarray analyses typically involves reverse transcribing RNA in a sample (starting RNA or input RNA) to generate double-stranded complementary DNA (cDNA), followed by an in vitro transcription reaction using the cDNA as a template to yield labeled complementary RNA (cRNA, also called antisense RNA). These protocols generally require microgram quantities of starting RNA to produce target cRNA for use in microarray analyses (Duggan et al. (1999) Nat. Genet. 21:10–14; Lockhart et al. (1996) Nat. Biotechnol. 14:1675–80; McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:13555–60; Schena et al. (1995) Science 270:467–70; Ramaswamy & Golub (2002) J. Clin. Oncol. 20:1932–1941). Thus, the applicability of these protocols is limited by the availability of sufficient quantities of starting RNA.
There are many situations in which either the total amount of RNA that can be obtained from a tissue sample is very low or in which there is a low copy number of expressed mRNA transcripts per cell. Thus, there is a need for methods for generating targets that can be used for microarray analyses from limited quantities of starting RNA.